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DB-ALM Protocol n° 118 : EpiSkinTM Skin Corrosivity Test
Skin Irritation and Corrosivity

The corrosivity potential of a chemical may be predicted by measurement of its cytotoxic effect, as
reflected in the MTT assay, on the EPISKIN T M reconstituted human epidermis. (Validation study
protocol)Objective & Application
TYPE OF TESTINGLEVEL OF ASSESSMENTPURPOSE OF TESTING:::

replacement
toxic potential, toxic potency, hazard identification
classification and labelling

The test method was granted regulatory approval as a replacement for the in vivo skin corrosivity test
(Method B.40 bis, EU 2000, 2008; OECD Test Guideline 431, OECD 2004) and it is used for hazard
identification and classification of corrosive potential in order to fulfil the regulatory requirements
concerning the Registration, Evaluation, Authorisation and Restriction of Chemicals (EU, 2008).
Furthermore, the test method is recommended for its use within the context of the sequential skincorrosivity testing strategy (OECD Test Guideline 404, OECD 2002 and Method B.4, EU 2004 and 2008).
The test allows the identification of corrosive chemical substances and mixtures and enables theidentification of non-corrosive substances and mixtures when supported by a weight-of-the-evidence
determination using other existing information (OECD, 2004 and EU, 2008).

Résumé
Most international regulatory classification schemes define chemically induced dermal corrosion as fullthickness destruction (necrosis) of the skin tissue, while some extend the definition of corrosion to
include any irreversible alterations caused to the skin.
The potential to induce skin corrosion is an important consideration in establishing procedures for thesafe handling, packing and transport of chemicals.
The determination of the skin corrosion potential is therefore included in international regulatory
requirements for the testing of chemicals, such as the U.S. Code of Federal Regulations (US DOT, 1991),
the updated OECD Test Guideline No 404 (OECD, 2002) and the Method B.4 of the Annex to Commission
Regulation 440/2008/EC (EU, 2008). Corrosivity was usually determined in vivo using the Draize rabbit
skin test (Draize et al., 1944).
The present test is based on the experience that corrosive chemicals show cytotoxic effects following
short-term exposure of the stratum corneum of the epidermis.
The test is designed to predict and classify the skin corrosivity potential of a chemical by assessment of
its effect on a reconstituted human epidermis.
EPISKIN Standard Model™ is a three-dimensional human skin model comprising a reconstructedepidermis with a functional stratum corneum. Its use for skin corrosivity testing involves topicalapplication of test materials to the surface of the skin, and the subsequent assessment of their effects oncell viability. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity
measured by formazan production from MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide] (Fentem et al., 1998 and Fentem, 1999).

Experimental Description
Endpoint and Endpoint Measurement:
CELL VIABILITY: Cell viability as determined by reduction of mitochondrial dehydrogenase activity
measured by formazan production from MTT
Endpoint Value:

Experimental System(s):RHE EpiSkin T M : Small Model (EpiSkinT M -SM, EPISKIN SNC Lyon, France) is a three-dimensional
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consisting of a collagen type I matrix coated with type IV collagen. A highly differentiated and stratified
epidermis model is obtained after 13-day culture period comprising the main basal, supra basal,
spinous and granular layers and a functional stratum corneum (Tinois et al., 1994).
Basic Procedure
Test materials are applied to the stratum corneum of the epidermal model (one epidermis unit per test
material) for three different exposure periods: 3 minutes, 1 hour, and 4 hours. Exposure to the test
chemical was terminated by rinsing with phosphate buffered saline (PBS). EPISKIN™ cultures exposedto the control compounds for 240 min serve as the controls for all three exposure periods.For each test
material, three independent tests with three different batches of EPISKIN™ are to be undertaken.
The viability of the epidermis is assessed by measuring the mitochondrial activity. The tissues are
incubated for 3 hours with MTT solution (0.3 mg/l; 2.2 ml per well). MTT, a yellow-coloured tetrazolium
salt, is reduced by succinate dehydrogenase into a blue formazan precipitate in the mitochondria ofliving cells. The precipitated formazan is extracted overnight by using acidified isopropanol (0.85 ml),and is then quantified spectrophotometrically at a wavelength between 545nm and 595nm. All
experimental procedures have to be conducted at room temperature (18-28°C); if the temperature is
below 20°C, the 3 hours MTT incubation should be carried out in a warmer environment of 20-28°C.
NaCl (50 µl) and glacial acetic acid (50 µl) are used as negative and positive controls, respectively.
Some highly reactive chemicals can produce fumes which may affect adjacent units in the same plate. It
is recommended that if there is any suspicion that a material could cause fumes, it should be tested alone
in a single plate. It is particularly important that the negative control units are not exposed to fumes fromother units, hence it is recommended to routinely incubate positive and negative controls in a separate
plate.

Data Analysis/Prediction Model
The test results are interpreted on the basis of the exposure time needed to cause cell viability todecrease below 35%.
The determination of the UN packing groups and EU classifications is summarized in the table reported
in the section 4.1. "Interpretation of test results" of the present standard operating procedure.

Test Compounds and Results Summary
A total of 60 test compounds, consisting of 11 organic acids, 10 organic bases, 9 neutral organics, 5phenols, 7 inorganic acids, 4 inorganic bases, 3 inorganic salts, 8 electrophiles, 3 soaps/surfactants(Barratt et al., 1998) have been tested in the ECVAM validation study.

StatusParticipation in Evaluation Studies:
An in-house evaluation and prevalidation study of the test method protocol was carried out during
1994-1996 (ECVAM, 1996).
Participation in Validation Studies:
During 1996 and 1997, this method has been evaluated in the ECVAM International Validation Studyon In Vitro Tests for Skin Corrosivity (Barratt et al., 1998 and Fentem et al., 1998). Based on theoutcome of the study, the ECVAM Scientific Advisory Committee concluded that the test was able todistinguish between corrosive and non-corrosive chemicals for all of the chemical types studied; it wasalso able to distinguish between known R35 (UN packing group I) and R34 (UN packing groups II & III)chemicals.
ESAC unanimously endorsed the statement that the EPISKIN™ test was scientifically validated for use asa replacement for the animal test and that this test was ready to be considered for regulatory acceptance(ESAC, 1998).
Regulatory Acceptance:
In 2000, the human skin model assays, which meet certain criteria (such as EPISKIN™ and
EPIDERM™ ), have been included into "Annex V. Part B.40 on Skin Corrosion" of the "Directive
67/548/EEC on the Classification, Packaging and Labelling of Dangerous Substances" (EU, 2000).
The test method was further recommended to be used as one of the in vitro methods for skin corrosivity
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testing in the OECD Test Guideline 404 (OECD, 2002) and in the Method B.4 of Annex V of the Directive
67/548/EEC (EU, 2004) laying down the step-wise testing strategy for classifying skin corrosives by thesequential application of three alternative methods: structure-activity relationships, pH measurements
and a single in vitro method (Worth et al., 1998).
In 2004, In Vitro Skin Corrosion: Human Skin Model Test was adopted as the OECD Test Guideline No
431 which is applicable to the assays for skin corrosion employing reconstituted human skin
(EPISKIN™ and EPIDERM™) models (OECD, 2004).
In May 2008, all the test methods of Annex V of the Directive 67/548/EEC have been incorporated into
the Annex to the Commission Regulation 440/2008/EC, laying down test methods pursuant toRegulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration,
Evaluation, Authorisation and Restriction of Chemicals (REACH) (EU, 2008).
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PROCEDURE DETAILS, March 1997*
EpiSkinTM Skin Corrosivity Test
DB-ALM Protocol n° 118

this protocol presents the standard operating procedure used in the ECVAM International
Validation Study on In Vitro Tests for Skin Corrosivity (Fentem et al., 1998).
* The accuracy of the SOP has been confirmed in October 2000.
During the preparation of the regulatory test guidelines some refinements have been
introduced into the test method. Therefore, the proposed update of the SOP has been sent
to the person responsible for the method for review and can be provided on request. As
soon as new information will become available this version of the protocol will be updated.

Contact Details
Dr. David J. Esdaile
Scientific Director
LAB Research - Hungary
Veszprem
Szabadsagpuszta H-8200
email: david.esdaile@labresearch.hu
telephone: +36 88 545-242

1. Introduction
Product Description
The EPISKIN-SM™ (Standard Model) kit contains 12 reconstructedepidermis units. Each reconstructed epidermis unit consists of a humancollagen (Types III and I) matrix, representing the dermis, covered with afilm of Type IV human collagen, upon which stratified differentiatedepidermis derived from human keratinocytes has been laid. Test
materials can be applied directly to the stratum corneum.
Precautions
The epidermal cells are taken from healthy volunteer donors negative toanti-HIV 1 and 2, and to hepatitis C, antibodies, and to hepatitis B
antigens. Nevertheless, normal handling procedures for biologicalmaterials should be followed:(a) it is recommended that gloves are worn during handling;and(b) after use, the epidermis, the material in contact with it,
and the culture medium, should be decontaminated (forexample, by using a 10% solution of bleach or a 1% solutionof pyosynthene), prior to disposal.
Quality Control
EPISKIN-SM kits are manufactured according to defined quality
assurance procedures (certified ISO 9001). All biological components ofthe epidermis and the kit culture medium have been tested for thepresence of viruses, bacteria and mycoplasma. The quality of the final
product is assessed by undertaking an MTT cell viability test and acytotoxicity test with sodium dodecylsulphate (SDS).
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cytotoxicity test with sodium dodecylsulphate (SDS).
For reasons connected with the nature of the product, it is shipped
before all of the necessary checks have been completed. A release form
certifying the conformity (or otherwise) of the batch is sent to the user, byfax, on the day of delivery of the kit.

2. Materials
2.1. KIT CONTENTSDESCRIPTION

USE

1 EPISKIN-SM plate containing 12
reconstructed epidermis units(area: 0.38cm 2)each reconstructed epidermis is attached tothe base of a tissue culture vessel with an
O-ring set and maintained on nutritive agar
for transport

1 12-well assay plate

for assays

1 flask of sterile assay medium

basic medium for use in assays

1 EPISKIN-SM biopsy punchfor easy sampling of epidermis

1 lot of "MTT reagents" :1 flask MTT reagent

to reconstitute

1 flask PBS 10x wash solution

to dilute

1 flask 4N NaOH

to adjust pH of wash solution

1 flask extraction solution of
isopropanol acid (ready to use)

1 flask negative control (NaCl, 9g/l)

specific controls for the corrosivity test

1 flask positive control(glacial acetic acid)

2.2 MATERIALS NOT PROVIDED WITH THE KIT
500ml wash bottle
5ml glass tubes with corks
200µl micropipetteMultidispenser micropipette (2.2ml)
50µl or 100µl positive displacement micropipette(for applying thick or viscous samples)
Vacuum source and Pasteur pipettes
Small forceps
TimersMicroplate reader with filter of 545-595nm and 96-well
microplates;or spectrophotometer and 1ml microcells
Vortex mixer
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Non-sterile ventilated cabinet

3. Experimental Procedures and Timing
Details of the kit and assay procedures should be registered on thereporting form (Annex 1).

3.1 RECEIPT OF TEST KIT
Check the date of dispatch written on the package. Before opening the
EPISKIN-SM kit:a) inspect the colour of the agar medium used for transportand check that its pH is acceptable: orange colour = good;yellow or violet colour = not acceptable;
andb) inspect the colour of the temperature indicator to verify
that the kit has not been exposed to a temperature above
40°C: the indicator changes from white to grey at 40°C.
In the event of any anomaly, immediately contact the Sales
Administration Department at SADUC (Tel: +33 78 56 72 72; Fax: +33 78
56 00 48).
Place the assay medium supplied with the kits at 2-8°C. Leave the
EPISKIN-SM kits in their packaging at room temperature until the assays
are to be undertaken.

3.2 APPLICATION AND RINSING
Safety precautions: MTT and corrosive materials are dangerous. Work ina non-sterile, ventilated, cabinet, wear protective gloves, and a mask andsafety glasses, as necessary.
Pre-warm the assay medium to 37°C. An approximate timing for
conducting the test procedure is given below as a guide.
9.30: proceed with the application of test material for the 4 hours samples(a) Fill the appropriate number of wells of an assay platewith pre-warmed culture medium (2.2ml per well). Markthe plate lids with the application time (4 hours) and thecode numbers of the chemicals to be tested (1 well per
chemical), or negative control (3 wells) or positive control(3 wells).
(b) Open the EPISKIN-SM kits and place an epidermis unit
into each prepared well. Mark each epidermis unit with theappropriate code number.
9.45: application of the products during 4 hours:(c) Add 50µl of test material to each well by using thepositive displacement pipette.
(d) In the case of solids, the material should be crushed to a
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page 6 / 15© ECVAM DB-ALM: Protocol(d) In the case of solids, the material should be crushed to a
powder, if necessary, and 20mg applied evenly to theepidermal surface (with difficult materials, use sufficient tocover the epidermal surface); add 100µl NaCl (9g/l saline) toensure good contact with the epidermis.
(e) Add 50µl NaCl (9g/l saline) to each of the three negative
control wells.
(f) Add 50µl glacial acetic acid to each of the three positive
control wells.
(g) Replace the lid on the plate and incubate for 4 hours(± 5 minutes) in a ventilated cabinet at room temperature(18-28°C).
Note:Tthe negative and positive controls incubated for 4 hours will act ascontrols for all of the incubation times.
10.00: proceed with the application of test material for the 1 hour samples(a) Fill the appropriate number of wells of an assay platewith pre-warmed culture medium (2.2ml per well). Markthe plate lids with the application time (1 hour) and thecode numbers of the chemicals to be tested (1 well per
chemical).
(b) Open the EPISKIN-SM kits and place an epidermis unit
into each prepared well. Mark each epidermis unit with theappropriate code number.
10.15: application of the products during 1 hour(c) Add 50µl of test material to each well by using thepositive displacement pipette.
(d) In the case of solids, apply 20mg and add 100µl of NaCl
(9g/l), as described previously for the 4 hours samples.
(e) Replace the lid on the plate and incubate for 1 hour (± 5minutes) in a ventilated cabinet at room temperature(18-28°C).
10.30: proceed with the application of test material for the 3 minutesamples(a) Prepare the MTT solution (0.3mg/ml; enough for 2.2ml
per well for the entire assay) and the PBS 1x wash solution,
as indicated in the "MTT reagents" leaflet accompanying
the test kit.
(b) Fill the appropriate number of wells of an assay platewith pre-warmed culture medium (2.2ml per well). Markthe plate lids with the application time (3 minutes) and thecode numbers of the chemicals to be tested (1 well per
chemical).
(c) Open the EPISKIN-SM kits and place an epidermis unit
into each prepared well. Mark each epidermis unit with theappropriate code number.

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10.45: application of the products during 3 minutes(d) Add 50µl of test material to each well by using thepositive displacement pipette. Proceed well-by-well at
20-second intervals, with the aid of multiple timers (test amaximum of 5 or 6 materials at a time). Ensure that the
exposure period is exactly 3 minutes for each well(e) In the case of solids, apply 20mg and add 100µl of NaCl
(9g/l), as described previouslyfor the 4 hours samples.
(f) Remove the EPISKIN-SM unit and rinse thoroughly with
PBS 1x solution, to remove all of the test material from theepidermal surface.
(g) Replace the EPISKIN-SM unit in the culture medium.
(h) When all of the units have been rinsed:
remove the culture medium
place the units on absorbant paper, or remove therest of the PBS from the epidermal surface with a
Pasteur pipette linked to a vacuum source (becareful not to touch the epidermis
add 2.2ml of the MTT solution (0.3mg/ml) to eachwellreplace the lid on the plate. If the ambient
temperature is 20-28°C, leave to incubate for 3
hours (± 5 minutes) in a ventilated cabinet at room
temperature, protected from light. If the ambient
temperature is below 20°C, then leave to incubate
for 3 hours (+ 5 minutes) at temperature of
20-28°C, protected from light. An incubator (withor without CO2), or a warm location within thelaboratory, may be used. It is important that all thesamples from each exposure time are treated
identically.
11.15: rinse the 1 hour samples and replace the culture medium with
2.2ml of MTT solution (0.3mg/ml), as described above.
11.45: place 0.85ml of acidified isopropanol into labelled glass tubes (onetube corresponding to one well of the tissue culture plate). Label eachtube with the name of the test material and the incubation time.
13.45: rinse the 4 hours samples and replace the culture medium with
2.2ml of MTT solution (0.3mg/ml), as described above.

3.3 FORMAZAN EXTRACTION
At the end of each incubation with MTT (14.15, 14.45 and 17.00), theformazan extraction should be undertaken:(a) place the units on absorbant paper(b) remove the MTT solution from each well(c) take a biopsy of the epidermis by using the biopsy punch,by placing the epidermis unit on the plate lid(d) separate the epidermis from the collagen matrix with thehttps://ecvam-dbalm.jrc.ec.europa.eu/methods-and-protocols/protocols

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aid of forceps, and place both parts (epidermis and collagenmatrix) into the acidified isopropanol(e) cork each tube and mix thoroughly by using a vortex
mixer(f) ensure that the acidified isopropanol is in good contact
with all of the material(g) store at room temperature overnight, protected fromlight.

3.4 ABSORBANCE/OPTICAL DENSITY MEASUREMENTS
Following the formazan extraction (left overnight):(a) mix each tube by using a vortex mixer(b) let the solution settle for 1-2 minutes, so that any cell fragments do
not interfere with the absorbance readings(c) place a 200µl sample from each tube into the wells of a 96-well plate(labelled appropriately)(d) read the optical densities (OD) of the samples at a wavelengh between
545nm and 595nm using acidified isopropanol solution as the blank.
(e) record the results on the template given in Annex 2.
NB. if a spectrophotometer is used rather than a plate reader, place a
500µl sample from each tube and 500µl isopropanol (not acidified) in a
1ml microcell and read the OD at 545-595nm using the acidified
isopropanol solution as the blank.

4. Calculations of Viability Percentages and Acceptability Criteria
Record all calculations on the Data Report Form (Annex 3).
Viability (%) = 100 x (OD test material/mean OD negative control at 4hours)(a) calculate the mean OD of the 3 negative control values: thiscorresponds to 100% viability. Based on historical data the minimum
acceptable mean OD for negative controls is 0.115 (mean ± 2SD). The
maximum acceptable mean OD for the negative control is 0.4 (to allow
for incubations at 28° C).
(b) calculate the mean OD of the 3 positive control values: the % viabilityof the positive control is calculated relative to the mean negative control.
Based on historical data (mean ± 2SD), the acceptable mean percentage
viability range for positive controls is 0-20%.
(c) calculate the % viability following exposure to the test material ateach incubation time as the OD expressed as a percentage of the mean
negative control value.
(d) assay acceptability criteria: for an assay to be acceptable, the mean
positive and negative control values should fall within the ranges given
above. In those cases where the mean values fall outside the range, theassay should be repeated, except in cases where the same chemical has
been tested on at least two other occasions (with acceptable control
values) and the results of all of the tests give the same corrosivity
classification.
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classification.
4.1 INTERPRETATION OF TEST RESULTS
The test results are interpreted on the basis of the exposure time needed tocause cell viability to decrease below 35%.The determination of the packing
group is summarized in the following table:

Classification
Packing group
UN

EU

Criteria for In Vitro interpretation

Corrosive class I

If viability < 35% after 3 min exposure

Corrosive class II

If viability >= 35% after 3 min exposureand < 35% after 1 hour exposure

Corrosive class III

If viability >= 35% after 1 hour exposureand < 35% after 4 hours exposure

Non corrosive

If viability >= 35% after 4 hours exposure

Corrosive class R35

If viability < 35% after 3 min exposure

Corrosive class R34

If viability >= 35% after 3 min exposureand < 35% after 4 hours exposure

Non corrosive

If viability >= 35% after 4 hours exposure

In cases where the viability values from individual skin units are highlyvariable, causing different corrosivity classifications, the chemical should
normally be retested. If one or more sets of data are considered to beincorrect (or inconsistent with data from other runs), the results should
be replaced by those generated in a repeat run.
In cases where the viability values fall below 35% but longer exposure
times give values of >35% (or values higher than the earlier time point),
the results should be considered to be doubtful. The run should normallybe repeated.
After having undertaken three independent tests with three different
batches of EPISKIN-SM™ for each test material, the Data Compilation
Form (Annex 4) should be completed.

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ANNEX 1

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ANNEX 4

Bibliography
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ECVAM (1996)
ECVAM News and Views.
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Alternatives to Laboratory Animals (ATLA) 24, 653-655
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and Liebsch M. (1998)
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The Episkin model: successful reconstruction of human epidermis in vitro.
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An evaluation of the proposed OECD testing strategy for skin corrosion.
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